Technical Abstract:
Pyrenophora teres, the causal agent of net blotch of barley survives as seedborne mycelium or pseudothesia in infested host residues. Besides the common netlike and occasional spot form symptoms on barley leaves, diffused dark or pale symptoms which are difficult to distinguish from other fungi are produced on kernels. Examination of conidia is considered the most accurate way to diagnose net blotch of barley. We developed a PCR technique to detect P. teres in conidia and seeds. In this technique, conidia and barley seeds showing symptoms were first homogenized in Extract-N-Amp Plant PCR Kit (Sigma-Aldrich) extraction solution and diluted with another solution from the kit to sidestep standard DNA extraction. Freeze dried mycelial cultures from P. teres f. teres and P. teres f. maculata were treated as seeds and conidia to serve as controls. Aliquots of the homogenate were added to PCR reaction and subjected to amplification using P. teres actin based PTACTIN980 and ITS primers. Sizes of amplicons from infested seeds and conidia which were resolved on agarose gel correlated with amplicons from the control P. teres cultures. The amplicons, purified from gels, sequenced and compared by alignment confirmed the detection of P. teres. The technique will accelerate positive detection of P. teres in infested barley seeds from diseased kennels by positively distinguishing it from other fungi and hasten the diagnosis from conidia.